1. Field of the Invention
The present invention relates to a process for producing human proinsulin in Escherichia coli (E. coli) using gene manipulation technology. More specifically, the present invention relates to a process for producing human proinsulin in a high yield by a novel expression vector having strong regulatory elements for the proinsulin gene and capable of producing a stable recombinant gene product.
2. Description of the Prior Art
The synthesis of human insulin using gene manipulation technology has been accomplished by one of the following two methods. In the first method, each gene of the alpha and beta chains of insulin is cloned and expressed. The proteins were purified followed by refolding them into insulin (Goeddel et al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76: 106-110; Chance et al., (1981) Diabetes Care, 4:149-154). However, this method has serious defects because separate preparation of two chains imposes undue tasks to those skilled in the art, and reconstitution of the two chains results in a significant decrease in yields. In addition, the reconstitution procedures are very complicated.
The second method is a direct production of insulin which comprises cloning a gene encoding the alpha and beta chains fused with another protein gene in a plasmid to produce the proinsulin fusion protein in bacteria analogous to the process by which insulin is secreted in the pancreas (William et al., (1982) Science, 215:687-689; Frank et al., (1981) in Peptides: Synthesis, Structure and Function; Proceedings of the Seventh American Peptide Symposium, Rich, D. H. and Gross, E., eds., Pierce Chemical Co., Rockford, Ill., pp. 729-738). This method is useful since it requires a single fermentation and a simple isolation procedure to obtain the proinsulin. In addition, the proinsulin can be refolded into tertiary structure more efficiently as compared to the first method.
However, since the yield of foreign protein such as proinsulin in intracellular expression in E. coli by gene manipulation technology is inversely proportional to the size of the expressed fused peptide, the insertion of a huge size of fused peptide gene into the recombinant expression vector of the proinsulin gene is undesirable. Thus, in order to insure stability of proinsulin in E. coli and simplify the purification procedure, there is a need to design a fused gene in which both facts are taken into consideration. Over the past decade, the present inventors and other research groups have attempted to reduce the size of the fused peptide by removing the .beta.-galactosidase portion (Guo et al., (1984) Gene 9:251-255; Yoon et al., (1988) In: Recombinant DNA Techniques, J. W. Yoon, editor, KOSCO Inc. Seoul, pp. 93-115), or replacing this peptide with a short fused peptide (Sung et al., (1986) Proc. Natl. Acad. Sci. U.S.A. 83:561-565). In addition, an attempt to maintain intracellular stability in E. coli of proinsulin expressed by combination of multiple proinsulin genes has been made, and E. coli which is deficient in a specific cellular protease has been used as a host to protect the intracellular degradation of proinsulin.
As a result, minor improvements have been made, but many limitations still exist, including a) problems in reducing the size of gene to be fused; b) long expression time; c) difficulties in exact refolding of the modified proinsulin in vitro; and d) low yields.
In order to eliminate and minimize these drawbacks, the present inventors have constructed a new recombinant expression vector to produce proinsulin (pYK10-9). The inventors have found that it is possible to prevent the expressed proinsulin from being degraded in the cell and to escape from the target of protease by attaching an oligonucleotide (SEQ ID NO. 1) containing a gene encoding (Thr).sub.6 (SEQ ID NO. 2) to the 5'-end of the proinsulin gene, and efficiently control the expression of the fused proinsulin by using a novel plasmid into which a strong promoter (lambda P.sub.R) in combination with a proinsulin fusion protein gene containing a lac ribosome binding site are inserted.
However, although it was possible to increase the intracellular stability of proinsulin fusion protein by decreasing the size of the fused peptide, some problems still remain in producing insulin products on a large scale. That is, the administration amount of insulin currently used as a diabetic treating agent is considerably large, being 40 mg per dose. In addition, once the insulin is injected, it should be permanently administered. Therefore, the expression utilizing E. coli should be significantly enhanced because of the difficulty in the purification procedure.
Various methods, for example, using a strong promoter such as lambda P.sub.R promoter which the present inventors have used, using a synthetic promoter such as tac promoter, or inserting two or more genes under the control of single promoter, and the like, have been proposed to improve the expression.
However, the use of a single promoter has some limitations. Thus, a case wherein a plurality of genes are successively inserted under the control of a single promoter may be taken into consideration, but it is also difficult to use this alternative due to the following problems. First, when multiple structural genes are successively arranged under the single promoter, multimers to which a single protein is one dimensionally bound are produced. Thus, in order to make the protein to be an active monomer, an additional treatment, such as CNBr cleavage, should be carried out. However, this treatment is not so realizable in view of the fact that the reaction site of CNBr is methionine. Second, since a protein having a relatively large molecular weight is synthesized as compared with the monomer, substantial improvements in the expression cannot be expected. Furthermore, as with most critical problems, it has been found that physical control of gene expression, which is the fundamental requirement for gene expression as well as the development of industrial strains is impossible due to the absence of effective transcriptional and translational control means when each gene is expressed.
Therefore, to solve these problems, the present inventors have constructed a new recombinant expression vector to produce proinsulin (pYD21). As a result, the present inventors have found that it is possible to produce human proinsulin on an industrial scale by inserting two or more copies of a DNA expression cassette each comprising a lambda P.sub.R promoter, a proinsulin fusion protein gene, a lac ribosome binding site, and a phage fd transcription terminator into a single plasmid.